Abstract . Genetic manipulation of the human immune system is a powerful approach to combating infectious diseases and cancers. Clinically, the most common cell based therapeutic approach currently used is hematopoietic stem cell (HSC) transplantation. The possibility of stably introducing specific T cell receptors (TCRs) into stem cells to generate T lymphocytes targeting specific antigens of choice opens doors to new and exciting life-long vaccine and therapeutic strategies. Human embryonic stem cells (hESC) and the recently-described induced pluripotent stem (IPS) cells, have the potential to revolutionize transplant technology. The theoretical ability of these cells to differentiate into any cell type in the body opens the possibility of improved cell replacement, organ replacement or genetic therapies. Induced pluripotent stem cells have a particular attraction, in that they can theoretically be derived from individual patients and will thus express identical HLA molecules, so they would not be rejected by the patient's immune response. We have demonstrated that hESC can be induced to differentiate into cells of the T lineage, and that IPS cells also have hematopoietic potential. New genetic material introduced into these cells can be expressed throughout hematopoietic differentiation. Thus the potential for adaptation of these stem cells to clinical use is especially true in regards to genetic manipulation of the immune system. The possibility of using these totipotent stem cells has many advantages over the use of HSC, including ability to culture extensively and ease of genetic manipulation, however there are many gaps in our knowledge regarding control of differentiation into specialized mature cell types. Development of model systems to optimize and study hESC and IPS generation into functional T cells could be very helpful for advancing our ability to genetically manipulate immune responses. This proposal will focus on pre-clinical studies optimizing the generation, from totipotent stem cells, of human T lymphocytes directed to target human melanoma, by virtue of expression of TCRs specific for melanoma antigens. These studies will interact with other components in this Program Project to provide proof-of-principle that hESC and/or IPS can be used to manipulate the immune system to specifically target cancer by pursuing the following specific aims: 1) Develop melanoma-specific cytotoxic T cells from human embryonic and induced pluripotent stem cells ; 2) Determine how expression of MART-1 TCR influences thymopoietic potential of hESC and IPS cells; 3) Develop T-cell specific expression systems to optimize distribution of TCR expression in progeny of hESC and IPS. We hope that together with the other components of this Program Project we will provide important pre-clinical information that cancer-specific T cells can be generated from these versatile stem cells, and that this approach may have potential for treating melanoma.